Project Summary The studies proposed for this K08 application will use unique approaches to examine the effects of sphingolipid metabolism on the mediator of flow-induced vasodilation (FID) within the human vasculature. Recently it has been shown that ceramide, a bioactive sphingolipid known to be elevated in the plasma of patients with cardiovascular disease, is an independent predictor of major adverse cardiovascular events. A potential way ceramide could adversely affect outcomes is by its recently discovered effect on the mediator of FID. Arterioles exposed to ceramide dilate in response to flow by generating H2O2, a pro-inflammatory and pro- atherosclerotic mediator as opposed to nitric oxide (NO), the anti-inflammatory, anti-atherosclerotic mediator utilized by healthy adults in response to increased flow. On the contrary, sphingosine-1-phosphate (S1P), also within the sphingolipid family and a metabolite of ceramide, promotes NO-dependent FID. How sphingolipids affect vasoactive mediators formed during flow and impact vascular homeostasis remains largely unknown. The aims of this proposal examine how the balance of sphingolipids, also known as the `sphingolipid rheostat,' influences the generation of a specific FID mediator which will have a profound impact on the formation or prevention of inflammation and atherosclerosis. Overall this application represents the necessary first step to define the mechanistic role of sphingolipid metabolism during FID in the human microcirculation. The training plan during the award phase will expand my scientific skill set in four ways. First, I will expand my training and experience using an established human vascular reactivity model to investigate how sphingolipid metabolism and the S1P:ceramide ratio determine the mediator of FID. Second, during a sabbatical in the laboratory of Dr. Ruikang Wang, I will receive training in tissue imaging, specifically in vivo microvascular imaging, with the goal of adapting a new technology (CytoCam) to translate our findings to the whole human. I will also receive training from Dr. Andrew Morris, in how to measure sphingolipids from small human samples using liquid chromatography tandem mass spectrometry (LC MS/MS). This instrumentation is available at MCW but has not been adapted for sphingolipid measurements. Bringing this technology to MCW will greatly strengthen my research program and support others needing to quantify lipid mediators in small tissue samples. Third, I will complete formal coursework in biostatistics, grant writing, advanced mass spectrometry techniques, clinical trial design, and medical imaging. These courses were specifically chosen by me and my mentoring team to supplement my formal training, and will provide vital knowledge as I continue to develop into an independent investigator. The fourth component of this training plan involves taking on leadership roles, such as being a mentor to students, managing an affinity group consisted of clinicians and researchers, organizing symposia at national meetings, and serving on scientific advisory committees for the American Society of Anesthesiology. The first three of these components are already in progress.